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PubMed1991

Neutralization activity of some Coxsackie B3 antibodies is compromised by protein factor(s) secreted by Vero cells.

When various anti-Coxsackie B3 virus antibodies were examined for the neutralizing activity in cultures under liquid medium, some antibodies including monoclonal antibodies gave abnormally low titers in the neutralization test in Vero cells, in comparison with the other cells such as HeLa, FL, HEp-2, or primary monkey kidney cells. The neutralization titer of these antibodies was, however, similar in all these cells by plaque reduction assays under agar overlay, i.e., the above phenomenon was restricted to cultures under liquid medium. The reduced neutralization titer in Vero cells under liquid medium was found to be brought about by protease-sensitive factor(s) released by Vero cells, because (1) the addition of Vero cell culture fluid resulted in a marked reduction of neutralizing titer in primary monkey kidney cells, and (2) the activity of the Vero cell factor was destroyed by trypsin (20 micrograms/ml for 1 h). As three-day incubation of virus-antibody complex in Vero cell culture fluid resulted in a partial restoration of virus infectivity, the binding of antibody to virions appears to be competed by the Vero cell factor.

Animals↗

PubMed1989

Bivens arm virus: a new rhabdovirus isolated from Culicoides insignis in Florida and related to Tibrogargan virus of Australia.

During field studies in 1981 on the transmission of bluetongue viruses in ruminants in Florida, a virus was isolated from Culicoides insignis collected near water buffalo (Bubalus bubalis) recently imported from Trinidad. Electron microscopy showed that this isolate, for which the name Bivens Arm virus is proposed, has rhabdovirus morphology. Serologic comparisons were made with recognized rhabdoviruses from terrestrial vertebrates and hematophagous arthropods. Indirect fluorescent antibody, complement fixation and neutralization tests indicated antigenic reactivity between Bivens Arm virus and two rhabdoviruses found only in Australia, Tibrogargan and Coastal Plains viruses. The Australian isolates cause subclinical infections in cattle and water buffalo and are believed to be transmitted by Culicoides. Initially, it was thought that Bivens Arm virus may have been introduced to Florida with the water buffalo from Trinidad, but a serologic survey of cattle serum, collected before the importation of the buffalo revealed antibody to the virus in cattle on farms located in diverse areas of Florida.

Animals↗

PubMed1997

Comparative antibody response in harbour and grey seals naturally infected by a morbillivirus.

The antibody response of free-ranging harbour and grey seals, naturally infected by a morbillivirus, was assessed using a virus neutralizing test and a radio-immunoprecipitation assay. The prevalence of antibody was similar between species, however, grey seals had significantly higher virus neutralizing titers. Serum from clinically healthy grey seals precipitated the nucleocapsid (N) protein along with the hemagglutinin (H) and fusion (F) glycoproteins. By contrast, significantly fewer harbour seal sera precipitated the envelope glycoproteins and responses were weaker than those of grey seals. One harbour seal with acute morbillivirus pneumonia, and two with encephalitis precipitated only the N protein. Serum from four harbour seals with encephalitis weakly recognized the envelope glycoproteins. Thus, the antibody response of grey seals appears more competent than that of harbour seals with respect to morbillivirus antigens. We speculate that this difference between the species may be an important determinant of morbillivirus susceptibility.

Animals↗

PubMed2003

Development of a novel vaccinia-neutralization assay based on reporter-gene expression.

In anticipation of large-scale smallpox vaccination, clinical trials of new vaccine candidates with improved safety profiles, and new vaccinia immune globulin (VIG) products, there is an immediate need to develop new assays to measure vaccinia-specific immune responses. The classical assay to measure vaccinia neutralization, the plaque-reduction neutralization test (PRNT), is slow, labor intensive, and difficult to validate and transfer. Here we describe the development of a novel vaccinia-neutralization assay based on the expression of a reporter gene, beta-galactosidase (beta-Gal). Using a previously constructed vaccinia-beta-Gal recombinant virus, vSC56, we developed a neutralization assay that is rapid, sensitive, and reproducible. The readout is automated. We show that the neutralizing titers, ID(50), for several VIG products measured by our assay were similar to those obtained by PRNTs. A new Food and Drug Administration VIG standard was established for distribution to other laboratories. The new assay will serve as an important tool both for preclinical and clinical trials of new smallpox vaccines and for evaluation of therapeutic agents to treat vaccine-associated adverse reactions.

Animals↗

PubMed1980

Indirect enzyme-linked immunosorbent assay (ELISA) for the detection of Coxsackievirus group B antibodies.

An indirect, solid phase, microplate enzyme-linked immunosorbent assay (ELISA) was found to be highly sensitive and reliable for detecting antibodies to the group B Coxsackieviruses and other picornaviruses. Conditions for obtaining maximum sensitivity and reproducibility of the indirect ELISA are described. Antibody titres were comparable to those obtained by the virus neutralization test and over 50 times higher than those obtained by the complement-fixation test. Purified virions used in the indirect ELISA reacted with low levels of cross-reacting heterotypic antibodies elicited by each of the six group B Coxsackieviruses, although homotypic reactions resulted in highest titres.

Animals↗

PubMed2004

Neutralization assay using a modified vaccinia virus Ankara vector expressing the green fluorescent protein is a high-throughput method to monitor the humoral immune response against vaccinia virus.

Vaccination against smallpox is again considered in order to face a possible bioterrorist threat, but the nature and the level of the immune response needed to protect a person from smallpox after vaccination are not totally understood. Therefore, simple, rapid, and accurate assays to evaluate the immune response to vaccinia virus need to be developed. Neutralization assays are usually considered good predictors of vaccine efficacy and more informative with regard to protection than binding assays. Currently, the presence of neutralizing antibodies to vaccinia virus is measured using a plaque reduction neutralization test, but this method is time-consuming and labor-intensive and has a subjective readout. Here, we describe an innovative neutralization assay based on a modified vaccinia virus Ankara (MVA) vector expressing the green fluorescent protein (MVA-gfp). This MVA-gfp neutralization assay is rapid and sensitive and has a high-throughput potential. Thus, it is suitable to monitor the immune response and eventually the efficacy of a large campaign of vaccination against smallpox and to study the vector-specific immune response in clinical trials that use genetically engineered vaccinia viruses. Most importantly, application of the highly attenuated MVA eliminates the safety concern in using the replication-competent vaccinia virus in the standard clinical laboratory.

Animals↗

PubMedDate not supplied

Cross-protection between Tacaribe complex viruses. Presence of neutralizing antibodies against Junin virus (Argentine hemorrhagic fever) in guinea pigs infected with Tacaribe virus.

Cross-protection between Junin virus and five other Tacaribe complex viruses and the serological response of guinea pigs inoculated with Tacaribe virus are reported here. Previous infection with Tamiami or Pichinde viruses significantly delayed guinea pig deaths. A 58% survival rate was found among animals immunized with three doses of Amapari virus, while guinea pigs inoculated with one dose of Machupo or Tacaribe virus were fully protected against Junin virus. Neutralization tests performed in serum samples of guinea pigs immunized with five doses of Tacaribe virus showed that they developed monologous and heterologous neutralizing antibodies.

Animals↗

PubMed2005

Characterization of the neurotoxin produced by isolates associated with avian botulism.

Several varieties of birds are affected by type C botulism. We conducted neutralization tests of culture supernatants of isolates from cases of avian botulism. Whereas the toxin produced by isolates derived from mammalian botulism was neutralized only with type C antitoxin, the toxins of all isolates related to avian botulism were neutralized with both type C and D antitoxins. An analysis of nucleotide sequences with several strains revealed that the neurotoxin gene in the isolates from avian botulism comprises two thirds of the type C neurotoxin gene and one third of the type D neurotoxin gene. This indicates that the neurotoxin of avian isolates is a mosaic of type C and D neurotoxins. We prepared three sets of primers to differentiate the gene for the mosaic form from the conserved genes of type C and D neurotoxins. The results of polymerase chain reaction with these primers indicated that all avian botulism-related isolates and specimens possess the gene for the mosaic form of the neurotoxin. The toxins purified from avian and mammalian isolates exhibited the same degree of lethality in mice, but the former showed greater toxicity to chickens than the latter. These results indicate that the mosaic neurotoxin is probably a pathogenic agent causing some forms of avian botulism.

Amino Acid Sequence↗

PubMed2000

Phase II safety and immunogenicity study of live chikungunya virus vaccine TSI-GSD-218.

We conducted a phase II, randomized, double-blind, placebo-controlled, safety and immunogenicity study of a serially passaged, plaque-purified live chikungunya (CHIK) vaccine in 73 healthy adult volunteers. Fifty-nine volunteers were immunized one time subcutaneously with the CHIK vaccine and 14 were immunized with placebo (tissue culture fluid). Vaccinees were clinically evaluated intensively for one month, and had repeated blood draws for serological assays (50% plaque-reduction neutralization test) for one year. Except for transient arthralgia in five CHIK vaccinees, the number and severity of local and systemic reactions and abnormal laboratory tests after immunization were similar in CHIK vaccinees and placebo recipients. Fifty-seven (98%) of 58 evaluable CHIK vaccinees developed CHIK neutralizing antibody by day 28, and 85% of vaccinees remained seropositive at one year after immunization. No placebo recipients seroconverted. This promising live vaccine was safe, produced well-tolerated side effects, and was highly immunogenic.

Adult↗

PubMed1996

[Evaluation of sensitivity and specificity of confirmatory assays for lupus anticoagulant (LA) detection].

To evaluate the sensitivity and specificity of confirmatory assays for LA detection, we performed the platelet neutralization procedure (PNP), diluted Russell's viper venom time (dRVVT) confirmatory assay (the ratio of LA-screen and LA-confirm:S/C), and specific hexagonal phase neutralization test (StaLA) on, plasma samples 43 LA-positive in either the ratio of dilute APTT and APTT(dAPTT/APTT) or dRVVT(LA-screen). The sensitivity and the specificity were evaluated by 4 different analyses based on, 1) maximal plasma dilution, 2) correlation between the screening assays and these confirmatory assays, 3) ratio of LA-positive in the confirmatory assays, and 4) receiver operation characteristic (ROC) method. All 4 analyses showed that sensitivity was in the order of PNP > S/C > Staclot-LA. However, the order of the specificity evaluated by the ROC method was Staclot-LA > S/C > PNP. The APTT-based confirmatory assay, PNP can detect LA of the plasma samples that were all positive in the dAPTT/APTT ratio, while the LA-positive plasma samples in dRVVT-confirm were all positive in the dRVVT-screen assay. Our findings suggest that confirmatory assays should be based on the method giving an abnormal screening assay. Accordingly, we recommend the following combinations of screening and confirmatory assays in terms of specificity; 1) dAPTT/APTT ratio and Staclot-LA as the APTT-based assay, and 2) LA-screen and LA-confirm as the dRVVT-based assay.

Blood Coagulation Tests↗

PubMed1982

Enzyme-linked immunosorbent assay: a rapid reproducible test for the measurement of rabies antibody.

An enzyme-linked immunosorbent assay (ELISA) was developed for detecting IgG antibodies to the Pitman Moore strain of rabies virus in sera from subjects immunised wih HDCS vaccine. End-point titres of antibody were determined using a pocket calculator preprogrammed to analyse absorbence values of test sera and negative controls. The assay was highly reproducible, and very close agreement was achieved when the results were compared with those of the mouse neutralization test. Rabies enzyme-linked immunosorbent assay is easy to perform, the results are obtained within 48 hours, and because it employs inactivated virus, the test can be used safely without special containment facilities.

Antibodies, Viral↗

PubMed1994

Comparison of VP4 and VP7 of five murine rotavirus strains.

The nucleotide and deduced amino acid sequences of outer capsid proteins VP4 and VP7 of five murine rotavirus strains (EW, EB, EC, EL, EHP) were determined. Comparisons of the VP7 amino acid sequences of the five murine rotavirus strains with rotavirus strains representative of G serotypes 1-14 showed that the murine strains were highly homologous to one another and more closely related to strains representing G3 than to any other G type. Analysis of the VP4 amino acid sequences of the murine strains revealed the presence of at least two murine P types. Therefore, sequence analysis would predict that the murine rotaviruses are G3 or G3-like and represent at least two unique P types (tentatively P17 for EW, EB, EC, and EL and P18 for EHP). When we attempted to categorize the murine strains by serum neutralization tests, our results were less clear. Serum to two murine strains, EHP and EW, displayed one-way reactivity by focus-reduction neutralization assays using several prototype G3 strains. The G3 serotyping monoclonal antibody 159 failed to neutralize EHP and EW, while the G3-specific monoclonal YO-1E2 neutralized EHP, but not EW. A reassortant (A15) containing VP7 from EW and VP4 from RRV was neutralized by these two G3-specific monoclonal antibodies to a level 8- to 20-fold more than EW, but was still 64- to 250-fold less than either SA11 and RRV. These results suggest that VP4 can influence the antigenicity of VP7.

Amino Acid Sequence↗

PubMed1997

The expression of potency of neutralizing antibodies for interferons and other cytokines.

The occurrence of antibody formation in patients administered biologically active human proteins as biotherapy for different diseases emphasizes the importance of establishing a common method of reporting neutralizing antibody levels for such cytokines. For quantitative neutralization bioassays, the preferred expression of the neutralizing potency of an antiserum is a titer, that is, the dilution of serum that reduces 10 Laboratory Units (LU)/ml of the cytokine to 1 Laboratory Unit/ml, the endpoint of most bioassays. This 10-to-1 LU/ml expression, which has been recommended by the World Health Organization for recording the results of interferon neutralization by the constant interferon method (with varying dilutions of serum) can also be used with the constant antibody method (with varying concentrations of interferon). For various reasons, interferon doses in International Units (IU)/ml should not be used for the neutralization test. Should the interferon concentration vary, intentionally or otherwise, from the intended dose of 10 LU/ml, a simple calculation allows expression of the neutralizing potency as the recommended reduction of 10-to-1 LU/ml as follows: the titer to be reported is the reciprocal of the antibody dilution (achieving the endpoint), multiplied by the interferon concentration (measured in that day's titration) minus one, divided by 9. This index of neutralization is the preferred method to represent the neutralizing potency of polyclonal and monoclonal antibodies and should make the results from different laboratories more readily interpretable and enable comparison.

Antibodies↗

PubMed2004

Probiotic bacteria stimulate virus-specific neutralizing antibodies following a booster polio vaccination.

BACKGROUND: Orally ingested probiotic bacteria may modulate the immune response and increase antibody titers against enteric infections by bacteria or viruses. Even though positive effects of probiotics on respiratory tract infections have been reported, overall only few studies have examined effects on virus infections concerning organs other than the gastrointestinal tract. AIM OF THE STUDY: It was the aim of the study to investigate whether and how probiotics affect the immune response to a standardized enterovirus challenge (polio) and infections not limited to the gastrointestinal tract in healthy adults. METHODS: In a randomized, controlled and double-blind study 64 volunteers consumed for 5 weeks chemically acidified clotted milk without bacteria or with 10(10)/serving (Lactobacillus rhamnosus ) GG or Lactobacillus acidophilus CRL431 added. In the second week subjects were vaccinated orally against polio 1, 2 and 3. Polio virus neutralizing serum activity, the primary parameter, was determined by the standard neutralization test (WHO) before and three times after vaccination. Polio-specific IgA, IgG and IgM were detected by ELISAs. RESULTS: Probiotics increased poliovirus neutralizing antibody titers (NT) and affected the formation of poliovirus-specific IgA and IgG in serum. The maximum increase after immunization was about 2, 2.2, or 4-fold higher, respectively, for NT, IgG or, IgA, in volunteers consuming probiotics instead of placebo. No consistent difference was noted between bacterial strains. CONCLUSIONS: Probiotics induce an immunologic response that may provide enhanced systemic protection of cells from virus infections by increasing production of virus neutralizing antibodies.

Adult↗

PubMed1977

A new method to detect lymphocytic choriomeningitis virus-specific antibody in human sera.

A plaque reduction method for measuring lymphocytic choriomeningitis virus-sensitizing antibody in human serum is described. One volume of virus and one volume of serially diluted human serum were mixed and incubated for 2 h at 37 degrees C. One volume of suitably diluted anti-human immune globulin antiserum was added and incubation continued for 0-5 h. Residual infectivity was then determined by means of a plaque assay employing L cell monolayer cultures and a methyl cellulose containing overlay medium. Of 75 sera from as many persons, 20 were positive with titres ranging from 640 to 10240. All positive sera were from verified cases or from persons who had had occupational contact with the virus. Close correlation of results was found between this method and a neutralization test employing mice, the one exception indicating that the in vitro assay for sensitizing antibody is more sensitive than the mouse assay for neutralizing antibody.

Animals↗

PubMed1986

Characterization of an echovirus type 30 variant isolated from patients with aseptic meningitis.

An outbreak of echovirus type 30 infection associated with aseptic meningitis occurred among newborn babies in a hospital neonatal room at Fukui City in 1983. The isolated virus was identified as an antigenic variant of echovirus type 30 by cross-neutralization tests with antisera against the prototype Bastianni strain and the present isolate. Western blot analysis demonstrated that the antigenic determinants responsible for virus neutralization, some of which were type specific and others strain specific, were present on the capsid protein VP1. The current strain was more thermoresistant than the prototype, suggesting some alterations in virus structural proteins.

Capsid↗

PubMedDate not supplied

[Testing of monoclonal antibodies to human interferon].

A comparative study of testing methods for polyclonal and monoclonal antibodies to human interferon using direct and reverse neutralization of the antiviral activity of interferon as well as ELISA was carried out. The activity of antibodies in ELISA was dozens of times higher than in neutralization tests. Polyclonal antibodies from the sera of mice immunized with alpha 2 interferon had a higher neutralizing capacity. M-5 monoclonal antibodies in specimens of ascitic fluid induced by inoculation of mice with hybrid cells exhibited an increase in both binding and neutralizing activity as compared with specimens of the culture fluid. Immunoglobulins from the ascitic and culture fluid of nonproductive myeloma cells as well as hybridomas producing monoclonal antibodies of other specificities showed practically no reaction with interferon in any of the tests under study. The screening of monoclonal antibodies intended for research and biotechnological purposes requires a composite analysis in both neutralization and binding tests in order to recover purposefully the hybrid clones producing antibodies with both or one of these properties.

Animals↗

PubMed1992

[The use of tapioca as coverage in viral titration].

Virus titration is an important step required on viral vaccines quality control. "Plaque assay", which employs several types of overlay media, is usually used on viral titrations. In this paper we describe the use of Tapioca as an overlay media. Firstly, the toxicity of Tapioca was tested on Vero cells inoculated or not with the Yellow Fever virus (YF) 17DD vaccine strain. Secondly, different batches of the 17DD virus using the Tapioca and Karaya gum as the overlay on Vero cells were tested when higher titres were obtained using Tapioca. Tapioca was also shown to be a suitable overlay to be used in thermostability and plaque reduction neutralization tests. Other systems could benefit from the use of Tapioca as an overlay, since it was possible to titer Measles virus in Vero cells. Tapioca is a cheap Brazilian product, is locally available, easy to use, and reliable. Its use is suggested.

Animals↗